Alpha-tocopheryloxyacetic acid (α-TEA) is a semi-synthetic derivative of naturally occurring vitamin E (alpha-tocopherol) that can be delivered via an oral route. Preclinical in vitro and in vivo data demonstrated that α-TEA is a potent anti-tumor agent with a safe toxicity profile in mice.
Guerrouahen et al BMC Cancer (2016) 16:199 DOI 10.1186/s12885-016-2220-6 RESEARCH ARTICLE Open Access GMP-grade α-TEA lysine salt: a 28-Day oral toxicity and toxicokinetic study with a 28-Day recovery period in Beagle dogs Bella S Guerrouahen1, Tobias Hahn2, Zefora Alderman2, Brendan Curti2, Walter Urba2 and Emmanuel T Akporiaye1,2* Abstract Background: Alpha-tocopheryloxyacetic acid (α-TEA) is a semi-synthetic derivative of naturally occurring vitamin E (alpha-tocopherol) that can be delivered via an oral route Preclinical in vitro and in vivo data demonstrated that α-TEA is a potent anti-tumor agent with a safe toxicity profile in mice We report a comprehensive study to evaluate the toxokinetics of good manufacturing practice (GMP)-grade α-TEA in dogs after daily oral administration for 28 days, followed by a 28-day recovery period Methods: Male and female beagle dogs received capsules of α-TEA Lysine Salt at doses of 100, 300, 1500 mg/kg/day α-TEA plasma levels were determined by high-performance liquid chromatography (HPLC) with mass spectrometric detection During the treatment, animals were observe for clinical signs, food consumption, body weight, and subjected to ophthalmoscopic, and electrocardiographic assessments At the end of the dosing period, blood was taken and toxicokinetic analyses and histopathology assessments were performed when animals were necropsied Results: Our findings showed that there was no α-TEA-related mortality or moribundity At the highest dose, increases in white blood cells and fibrinogen levels were observed These levels returned to normal at the end of the recovery period Histopathological evaluation of major organs revealed no significant lesions related to α-TEA-treatment Conclusion: We demonstrate that for designing clinical trials in patients, the highest non-severely toxic dose (HNSTD) of α-TEA is 1500 mg/kg/day in Beagle dogs and this data informed the design of dose-escalation studies of α-TEA in patients with advanced cancer Keywords: Cancer therapy, Vitamin analog, α-TEA lysine salt, Toxicokinetic, Pharmacokinetics, Non-rodent Background Alpha-tocopheryloxyacetic acid (α-TEA) is a semi-synthetic, non-hydrolysable ether derivative of vitamin E Structurally, α-TEA differs from vitamin E by the replacement of the hydroxyl group at the carbon number of the phenolic ring of the chroman head with an acetic acid residue linked by an ether bound [1] α-TEA is a cytotoxic drug that induces tumor cell death through targeting the mitochondria and by modulation of apoptosis and survival * Correspondence: eakporiaye@sidra.org Sidra Medical and Research Center, Experimental Biology Division – Research, PO Box 26999, Doha, Qatar Laboratory of Tumor Immunology and Therapeutics, Earle A Chiles Research Institute, Robert W Franz Cancer Research Center, Providence Portland Medical Center, 4805 NE Glisan St 2N35 Portland, OR, USA pathways [2] The in vivo anti-tumor activity of α-TEA has been reported in several pre-clinical tumor models [1], and is partially dependent on a T-cell-mediated immune response [3–6] Although α-TEA has been evaluated as an anti-cancer agent in numerous pre-clinical tumor models [1, 7–10], efforts to translate these findings into human clinical trials are lacking The goal of this study was to conduct a dose-escalation evaluation in an appropriate non-rodent animal species as required by the United States Food and Drug Administration (U S FDA) to obtain relevant toxico- and pharmaco-kinetic information in preparation for a first-inhuman trial to evaluate the safety and tolerability of αTEA in patients with advanced cancer α-TEA lysine salt © 2016 Guerrouahen et al Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 Methods tocopherol with ethyl bromoacetate to form the ethyl ether intermediate The ethyl ether intermediate was then reacted with potassium hydroxide to form α-TEA free acid The lysine salt was formed by adding aqueous lysine solution to a solution of α-TEA in isopropyl alcohol The lysine salt with its empirical formula of C37H66N2O6 and its molecular weight of 634.93 g/mol is a stable crystalline off-white powder The dose concentration analysis was performed during the study and the test material was stable Capsules for oral dose administration were prepared at least once weekly with appropriate amounts of bulk α-TEA placed into gelatin capsules (Pharmatek LLC, San Diego CA) Oral administration is the planned route of administration in humans The dose of the drug is expressed as free acid using a correction factor of 1.3 to reflect salt content (calculated as ratio of lysine salt/free acid molecular weight) Preparation of α –Tocopheryloxyacetic acid lysine salt in capsules Animal studies In the process of manufacturing α-TEA free acid for pre-clinical development, the contract Commercial Research Organization (Ricerca Biosciences, LLC, Concord OH) made the observation that during the initial scaleup, the free acid exhibited liquid crystal properties Therefore, a salt screen was performed, which led to the identification and selection of the lysine salt α-TEA lysine salt (α-TEA LS) was synthesized (Fig 1), using a modification of a previously described procedure [9] Briefly, α-TEA LS was prepared by reacting alpha-D- Initial studies in mice were conducted to assess the efficacy of the lysine salt form of α-TEA against established tumors Treatment of the animals (including but not limited to all husbandry, housing, and feeding conditions and euthanasia) was conducted in accordance with The Animal Welfare Act (Public Law 89-544) and the guidelines recommended in Guide for the Care and Use of Laboratory Animals (National Academy Press, Washington DC, 2011) The protocols and procedures involving the care and use of animals in was administered at increasing doses of 100, 300 and 1500 mg/kg to male and female beagle dogs for 28 consecutive days, and then observed for 28 days after the last α-TEA dose Complete measurements of body weight and food consumption were conducted over the treatment period Ophthalmologic and electrocardiographic observations were assessed and clinical pathology evaluated In this dose escalation study utilizing the lysine salt of αTEA, we demonstrated that daily administration of α-TEA was not toxic at 1500 mg/kg body weight The findings from this comprehensive and observational study to evaluate the pharmacology and toxicology of the αTEA (lysine salt) in beagle dogs formed the basis for initiating a first-in-human clinical trial in patients with advanced cancer that is ongoing (NCT 02192346) Fig Chemical structure of α-Tocopherol, α-Tocopheryloxy Acetic Acid (α-TEA), and α-tocopheryloxy acetic acid Lysine Salt (α-TEA LS) a α-Tocopherol Molecular Weight (MW) = 430.69 b α-Tocopheryloxy Acetic Acid (α-TEA) MW = 488 c α-TEA Lysine Salt MW = 634.93 Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 the study were reviewed and approved by the Earle A Chiles Research Institute and Ricerca Institutional Animal Care and Use Committees (IACUC) α-TEA was formulated in mouse chow and delivered to mice bearing established T1 mammary tumors, as we previously described This anti-tumor study was followed by a toxicology study For this purpose, both the α-TEA free acid and α-TEA LS forms were administered to naïve mice intravenously or by oral gavage A total of 44 Beagle dogs (Canis familiaris; were acquired by the contract CRO (Ricerca Biosciences) from Marshall BioResources, North Rose, NY) for the study Animals were as uniform in age as possible; dogs were pre-pubertal to young adults, at least months of age (range of 7.8 to 8.3 months of age) and weighed between and kg at the start of dose administration The frequency of administration was designed to mimic the regimen intended for human trial α-TEA LS was administered in gelatin capsules once daily to 22 male and 22 female dogs at 100, 300 and 1500 mg/kg for 28 consecutive days The amount for each animal was based on the most recent body weight Control animals received equal number of empty gelatin capsules as the high dose animals Animals were observed for viability and clinical signs, daily food consumption, body weight (once weekly or twice for high dose animals) Group assignment and dose level are shown in the Table Clinical pathology, ophthalmic and electrocardiography examinations were done at the pre-dose and after the terminal dose Eight dogs from each group (4 females and males) were necropsied at the end of the dosing period (Day 29) or at the end of the recovery period (Day 57) Animals were fasted overnight prior to scheduled blood test or necropsy When necropsied, organs were collected and preserved in 10 % neutral-buffered formalin with the exception of the testes, epididymides, and eyes; which were fixed in Modified Davidson’s Solution for 24 to 48 h, water washed, and then transferred to 10 % neutral-buffered formalin for storage Histopathology assessments were performed Bone marrow smears were stained with Wright-Giemsa before cytology evaluation Toxicokinetics studies Toxicokinetics studies were performed by Celerion Inc., a commercial research organization based in Lincoln, Nebraska, USA Toxicokinetic parameters were studied on blood collected from the jugular vein of the animal (1 mL) on day and on the last day of treatment Blood samples were collected into K3EDTA-containing tubes at 1, 2, 4, 8, and 24 h following the first α-TEA administration Following the last administration (day 28), blood collection for recovery animals was scheduled as follows: 2, 8, 24, 72, and 168 h For the control animals (group 1), only the 8-hour sample was collected The chosen time points were based on kinetic data gathered from pilot toxicity studies conducted in mice Recovered plasma was stored at -70 °C until analysis Data collection Animal data, such as observations, body weights, food consumption, clinical pathology values, necropsy findings, and organ weights, were collected and reported electronically using Provantis™, Version (Instem LSS Ltd Staffordshire, UK) Urine was collected and analyzed by a Clinitek Atlas Urinalysis System Data from the examination of urine sediment were entered directly into ProvantisTM Toxicokinetics data were collected and stored in electronic notebook system LabnotesTM Client 1.18 Sample preparation and High Performance Liquid Chromatography (HPLC) analysis Systemic levels of α-TEA were determined using HPLC with mass spectrometry Bioanalytical data were obtained from Celerion, Inc according to the SOPs written based on the GLP principles Briefly, an aliquot of the extracted dog plasma was analyzed by an HPLC equipped with an AB SCIEX API 4000TM triple quadrupole mass spectrometer using an ESI source Negative ions were monitored in the multiple reaction-monitoring (MRM) mode Quantitation was determined using a weighted linear regression analysis (1/concentration2) of peak area ratios of the analyte and internal standard The area under the plasma concentration-time curves, peak Table Group assignment and dose levels Group Number of animals (M/F) Test article Nominal dose level (mg/kg/day) Actual dose levela Number of animals for necropsy (M/F) Terminal (Day 29) Recovery (Day 57) 6/6 Vehicle 0 4/4 2/2 4/4 α-TEA 100 130 4/4 - 6/6 α-TEA 300 390 4/4 2/2 6/6 α-TEA 1500 1950 4/4 2/2 a Dose levels have been corrected for lysine salt using a correction factor of 1.3 A total of 44 Beagle dogs (22 males and 22 females) received α-TEA lysine salt at different concentrations (0, 100, 300, 1500 mg/kg) Eight dogs from each group (4 females and males) were necropsied at the end of dosing period (Day 29) or at the end of recovery period (Day 57) Guerrouahen et al BMC Cancer (2016) 16:199 plasma concentration, time to achieve peak plasma concentration, and plasma terminal half-life (AUC, Cmax, Tmax, and T1/2) were determined using WinNonlin Version 6.2 (Pharsight, Mountain View CA), operating as a validated software system Statistical analysis For comparative statistics, data through the Day 29 termination were evaluated using the Levene Test for homogeneity of variances and the Shapiro-Wilks Test for normality of distributions, with significance at p ≤ 0.05 Data determined to be homogeneous and of normal distribution were evaluated for analysis of variance (ANOVA) If the ANOVA verified significance at p ≤ 0.05, pairwise comparisons of each treated group with the control group were made using a parametric test, Dunnett t-test, to identify statistical differences (p ≤ 0.05) Data determined to be nonhomogeneous or of non normal distribution were evaluated using a Kruskal-Wallis Test for group factor significance If significance (p ≤ 0.05) existed between groups, Page of 16 a nonparametric test, Wilcoxon with Bonferroni-Holm, was used to compare treated groups to the control group Results Anti-tumor activity of α-TEA lysine salt We first tested the anti-tumor activity of GMPmanufactured α-TEA LS, by conducting experiments in BALB/c mice bearing established T1 mammary tumors as previously described [11] Tumor bearing mice received (i) control diet until days post-tumor cell injection, and then were switched to 0.39 % αTEA salt diet; or (ii) 390 mg/kg body weight α-TEA LS diet After 60 days of tumor monitoring, we found that α-TEA LS significantly inhibit tumor growth (Fig 2a) and prolonged overall survival compared to control diet (Fig 2b) Blood samples were collected from naïve mice/group and systemic levels of α-TEA were determined by HPLC with mass spectrometry detection in order to compare the pharmacokinetics and exposure of a single dose of α-TEA LS with that of the Fig Anti-tumor activity of α-TEA lysine salt a Effect of dietary delivery of α-TEA lysine salt on primary tumor growth BALB/c mice were injected with 5x104 T1 tumor cells in the right mammary fat pad When tumors became palpable (~5 days post tumor cell injection) mice received 390 mg/kg/day α-TEA lysine salt diet (equivalent to 300 mg/kg/day α-TEA) and tumor growth was monitored b Oral α-TEA lysine salt prolongs survival of mammary tumor-bearing mice BALB/c with established T1 mammary tumors (day 5, ~ 4-6 mm2) received oral α-TEA lysine salt diet (390 mg/kg bodyweight) or nutrient-matched control diet Mantel-Cox analysis of survival of n = mice per group Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 Fig Exposure of BALB/c mice to a single-dose of lysine salt or free acid α-TEA α-TEA free acid (100 mg/kg) and α-TEA lysine salt (200 mg/kg) were administered by intravenous injection or oral gavage Blood was collected from mice/group at each time point and analyzed for α-TEA levels by high performance liquid chromatography (HPLC) with mass spectrometric detection (LC-MS/MS) free acid The free acid could only be administered by gavage because it was toxic when administered by intravenous injection However, both routes of administration could be used with the lysine salt As shown in Fig and Table 2, when α-TEA was administered orally by gavage at the same dose level (200 mg/kg), exposure was higher with α-TEA LS compared to αTEA free acid The peak plasma concentration after administration (Cmax) was times higher with α-TEA LS compared to the α-TEA free acid (Fig 3) We observed a saturation phenomenon reflected by a slight decrease in Cmax following the increase in dose level from 100 mg/kg to 200 mg/kg of α-TEA free acid (Table 2) Our data indicate that the time to reach the peak plasma concentration after administration (Tmax) is h with α-TEA LS versus 24 h for α-TEA free acid The elimination half-lives was similar between 100 and 200 mg/kg α-TEA free acid and 200 mg/kg α-TEA lysine salt (54.6/50.2/54.6 h respectively) Repeat daily dosing of α-TEA Lysine Salt did not cause gross toxicity Several experiments were performed to evaluate the pharmacology and toxicology profile of repeated dosing of GMP-grade α-TEA LS in Beagle dogs, over a period of 28 days followed by a 28-day recovery period Dose levels were chosen based on available data in mice [11] and from a preliminary dog tolerability study Once daily, α-TEA salt was administered in gelatin capsules to male and female Beagle dogs at 100, 300 and 1500 mg/kg body weight for 28 consecutive days Clinical signs were limited to fecal changes (decreased/discolored) and emesis in 1500 mg/kg animals on several occasions following start of dose administration and continuing until the start of recovery period The decreased feces coincided with transient decreases in food consumption noted in several animals, and persisted upon continued dosing α-TEA effects on food consumption at 1500 mg/kg were also reflected in the slightly decreased in body weight gain in Table Effect of a single dose of α-TEA lysine salt or free acid on serum pharmacokinetics parameters Test article Dose level (mg/kg) Tmax (hr) T½ (hr) Cmax (ug/mL) AUC0-24 (hr*ug/mL) AUC0-∞ (hr/*ug/mL) α-TEA Free Acid 100 54.6 20.4 1,180 1,670 α-TEA Lysine Acid 200 54 29.4 1,680 2,470 α-TEA Free Acid 200 24 50.2 14.7 1,020 1,400 α-TEA free acid (100 mg/kg) and α-TEA lysine salt (200 mg/kg) were administered by intravenous injection or oral gavage in BALB/c mice Blood was collected from mice/group at each time point and analyzed for α-TEA levels by HPLC with mass spectrometric detection (LC-MS/MS) The peak plasma concentration of a drug after administration (Cmax), the time required to reach it (Tmax), the time required for the concentration of the drug to reach half of its original value (T1/2), the area under the plasma drug concentration-time curve (AUC) were determined using WinNonlin Version 6.2 software system Guerrouahen et al BMC Cancer (2016) 16:199 all 1500 mg/kg males during the second and third week of dose administration and in two of six females during the second week and all the 1500 mg/kg females during the third week of administration (Fig 4a, b) Individual body weight gain decreases were present at 300 and 100 mg/kg; however, overall group mean was comparable to controls and the changes were not considered toxicologically relevant During the recovery period, the body weight gain in control and α-TEA-treated animals was comparable (data not shown) There was no moribundity or mortality throughout the course of treatment There were no α-TEA-related ophthalmologic findings, but the 1500 mg/ kg group showed sinus bradycardia on electrocardiograms at day 27 for the males and day 26 for the females Page of 16 Clinical pathology (erythroid, leukocytes, coagulation parameters, serum chemistry and urinalysis) of α-TEAtreated animals At the dosing level of 100, 300, and 1500 mg/kg, no effect on erythroid or serum chemistry was observed On day 29, white blood cell counts (neutrophils and lymphocytes) and fibrinogen levels were slightly higher in animals that received 1500 mg/kg α-TEA LS (in of males and of females) compared to their pre-dose level (Tables and 4) Interestingly, at the end of the recovery period, no α-TEArelated changes in coagulation or leukocyte parameters were recorded (Tables 5) On Day 29, evaluation of serum chemistry parameters showed that the mean chloride concentration (CL), mean blood urea nitrogen concentration Fig Measure of body weights during 28-day dosing of α-TEA lysine salt Male (a) and female dogs (b) received indicated doses of α-TEA lysine salt daily Body weights were determined twice before the start of the treatment and every week following the start of the treatment Data points represent mean body weight ± SD Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 Table Mean blood cells and coagulation parameters in dogs 10 days prior to treatment Vehicle Males 100 mg/kg 300 mg/kg 1500 mg/kg Unit Mean SD N Mean SD N Mean SD N Mean SD N Red blood cells 6.315 0.485 6.418 0.316 6.523 0.211 6.74 0.416 M/ul Hemoglobin 13.98 1.13 13.83 0.71 14.43 0.48 14.8 1.21 g/L Hematocrit 43.18 3.51 43.08 1.54 44 1.5 45.23 3.2 % Mean corpuscular volume 63.38 0.97 67.18 1.55 67.52 2.46 67.1 1.42 fL Mean corpuscular hemoglobin 22.1 0.3 21.58 0.62 22.12 0.78 21.95 0.76 Mean corpuscular hemoglobin concentration 32.37 0.18 32.13 0.61 32.73 0.2 32.73 0.56 g/dL Reticulocytes 24.63 13.54 29.25 7.68 22.28 5.7 21.75 10 109/L Erythrocytes pg Leukocytes White blood cells 7.473 1.869 7.373 1.858 9.175 2.951 7.103 1.64 K/uL Neutrophils 1.207 1.602 3.9 1.37 5.428 2.489 3.943 1.249 K/uL Lymphocytes 2.473 0.55 2.69 0.506 2.638 0.788 2.445 0.445 K/uL Monocytes 0.512 0.275 0.54 0.083 0.668 0.413 0.375 0.121 K/uL Eosinophils 0.128 0.077 0.14 0.022 0.282 0.171 0.193 0.108 K/uL Basophils 0.093 0.051 0.063 0.021 0.09 0.029 0.082 0.018 K/uL Platelets 381.7 39.4 330.8 80.1 427.8 82.2 360 57.4 K/uL Mean platelet volume 12.38 1.52 14.28 2.15 12.62 1.17 13.53 0.9 fL Protrombin time 6.63 0.24 6.88 0.17 6.8 0.3 6.85 0.19 Seconds Thrombocytes Females Activated partial thromboplastin time 13.12 0.82 12.9 0.66 13.83 0.81 13.33 0.83 Seconds Fibrinogen 270.2 47.3 312.5 47.5 282 44.6 259.8 56.7 Mg/dL Red blood cells 6.562 0.322 6.788 0.297 6.49 0.417 6.59 0.344 M/ul Hemoglobin 14.62 0.8 15.43 0.77 14.12 1.52 14.7 0.79 g/L Hematocrit 45.15 2.42 46.98 2.33 43.78 3.88 45.22 2.26 % Erythrocytes Mean corpuscular volume 68.82 2.46 69.2 1.83 67.4 2.3 68.63 1.52 fL Mean corpuscular volume hemoglobin 22.3 0.58 22.75 0.83 21.73 1.12 22.32 0.61 Pg Mean corpuscular hemoglobin concentration 32.4 0.41 32.85 0.31 32.17 0.65 32.55 0.21 g/dL Reticulocytes 32.4 15.63 41.9 19.22 34.42 14.98 25.3 11.84 109/L White blood cells 8.123 1.412 8.54 1.517 8.418 2.205 8.655 1.246 K/uL Neutrophils 4.873 1.151 4.855 1.139 4.88 1.404 5.457 0.986 K/uL Lymphocytes 2.482 0.62 2.793 0.381 2.578 0.459 2.482 0.213 K/uL Leukocytes Monocytes 0.473 0.079 0.513 0.131 0.505 0.185 0.353 0.119 K/uL Eosinophils 0.15 0.14 0.138 0.019 0.313 0.324 0.197 0.072 K/uL Basophils 0.093 0.047 0.148 0.038 0.08 0.029 0.102 0.014 K/uL platelets 358.8 83 350.5 81.3 319.2 53.1 356.7 49.3 K/uL Mean platelet volume 13.23 2.08 13.13 1.27 13.43 1.24 12.85 1.05 fL Prothrombin time 6.9 0.32 7.03 0.25 6.73 0.36 6.97 0.34 Seconds Activated partial thromboplastin time 13.42 0.65 13.4 0.41 13.37 0.44 13.12 0.37 Seconds Fibrinogen 227 54.8 225.8 90.2 219.2 42.6 205.8 35.3 mg/dL Thrombocytes Male and female dogs received indicated doses of α-TEA lysine salt daily for 28 days Ten days before the treatment start, blood was collected from dogs (0, 300, 1500 mg/kg treatment groups) or dogs (100 mg/kg treatment group) and analyzed for blood cells and coagulation parameters Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 Table Mean blood cells and coagulation parameters in dogs 29 days after the start date Vehicle Males 100 mg/kg 300 mg/kg 1500 mg/kg Unit Mean SD N Mean SD N Mean SD N Mean SD N Red blood cells 6.238 0.841 6.438 0.598 6.21 0.143 6.418 0.437 M/ul Hemoglobin 14.55 1.91 14.55 1.1 6.21 0.143 6.418 0.437 g/L Hematocrit 42.1 6.09 42.18 3.27 41.05 1.04 41.7 2.79 % Mean corpuscular volume 67.45 1.1 65.63 1.51 66.08 2.16 65 1.75 fL Mean corpuscular hemoglobin 23.35 0.24 22.68 0.59 22.9 0.64 22.73 0.7 pg Mean corpuscular hemoglobin concentration 34.6 0.47 34.6 0.37 34.63 0.33 34.9 0.51 g/dL Reticulocytes 42.73 11.31 58.65 15.92 46 11.97 24.55 3.67 109/L Erythrocytes Leukocytes White blood cells 7.438 0.919 9.23 1.25 12.23 3.277 11.91 3.574 K/uL Neutrophils 4.183 0.937 4.805 0.658 7.438 3.162 7.193 2.464 K/uL Lymphocytes 2.593 0.442 3.438 0.597 3.56 0.326 3.655 0.792 K/uL Monocytes 0.43 0.153 0.683 0.089 0.825 0.26 0.565 0.14 K/uL Eosinophils 0.095 0.039 0.178 0.055 0.223 0.101 0.388 0.328 K/uL Basophils 0.098 0.078 0.1 0.018 0.138 0.038 0.08 0.026 K/uL platelets 259.8 64 281.8 44.7 369.5 87.3 262.3 75.2 K/uL Mean platelet volume 10.95 1.11 9.88 0.8 0.87 8.6 0.77 fL Protrombin time 6.78 0.05 7.08 0.19 6.85 0.17 6.9 0.24 Seconds Thrombocytes Females Activated partial thromboplastin time 12.68 1.16 12.7 0.84 13.5 0.88 13.05 1.28 Seconds Fibrinogen 254.5 42.7 288.5 18.9 289.5 85.4 414.5 89.9 mg/dL Red blood cells 6.33 0.241 6.895 0.563 6.72 0.306 6.773 0.383 M/ul Hemoglobin 14.85 16.43 1.7 15.55 1.1 15.78 0.8 g/L Hematocrit 43.33 3.14 47.68 4.73 45.48 3.24 45.98 2.05 % Erythrocytes Mean corpuscular volume 68.33 2.53 69.1 1.14 67.83 3.07 67.88 1.6 fL Mean corpuscular volume hemoglobin 23.45 0.71 23.83 0.55 23.15 23.3 0.55 pg Mean corpuscular hemoglobin concentration 34.25 0.47 69.3 23.65 72.85 24.88 45.75 23.65 g/dL Reticulocytes 34.25 0.47 34.5 0.24 34.23 0.35 34.3 0.46 109/L White blood cells 8.81 3.586 9.75 2.4 9.743 1.237 13.31 1.721 K/uL Neutrophils 5.293 3.009 4.928 1.273 5.32 1.312 8.193 1.654 K/uL Lymphocytes 2.693 0.318 3.575 0.702 3.358 0.543 3.985 0.625 K/uL Leukocytes Monocytes 0.548 0.218 0.495 0.071 0.648 0.083 0.665 0.281 K/uL Eosinophils 0.163 0.111 0.505 0.467 0.273 0.1 0.3 0.124 K/uL Basophils 0.078 0.022 0.175 0.027 0.105 0.019 0.123 0.051 K/uL platelets 275.8 62.7 301.3 31.9 294.3 79 340 72.2 K/uL Mean platelet volume 13.03 2.23 10.45 0.79 10.85 0.97 9.75 0.71 fL Prothrombin time 7.1 0.18 7.18 0.15 6.8 0.22 6.93 0.19 Seconds Activated partial thromboplastin time 12.23 0.72 12.68 0.92 13.48 0.4 12.45 0.47 Seconds Fibrinogen 206 11.1 178.5 16.3 251.5 68.3 275.8 58.8 mg/dL Thrombocytes Male and female dogs received indicated doses of α-TEA lysine salt daily for 28 days After 28 daily treatments, blood was collected from dogs per treatment group and analyzed for blood cells and coagulation parameters Guerrouahen et al BMC Cancer (2016) 16:199 Page of 16 Table Mean blood cells and coagulation parameters in dogs at the end of the recovery period (Day 57) Vehicle Mean Males 300 mg/kg N Mean 1500 mg/kg N Mean Unit N Erythrocytes Red blood cells 5.975 6.13 6.115 M/ul Hemoglobin 14 14.2 14.15 g/L Hematocrit 40 40.7 40.65 % Mean corpuscular volume 67 6.35 66.7 fL Mean corpuscular hemoglobin 23.4 23.15 23.2 pg Mean corpuscular hemoglobin concentration 34.9 34.85 34.8 g/dL Reticulocytes 34.15 78.3 59.92 109/L White blood cells 8.705 8.025 10.37 K/uL Neutrophils 5.275 4.53 6.14 K/uL Leukocytes Lymphocytes 2.7 2.645 3.055 K/uL Monocytes 0.34 0.225 0.385 K/uL Eosinophils 0.21 0.225 0.385 K/uL Basophils 0.125 0.125 0.125 K/uL 229 290.5 310 K/uL Thrombocytes platelets Mean platelet volume 11.7 9.65 9.55 fL Protrombin time 7.05 7.15 7.15 Seconds Activated partial thromboplastin time 13.05 13.75 12.65 Fibrinogen Females Seconds mg/dL Erythrocytes Red blood cells 7.41 6.775 6.455 M/ul Hemoglobin 17.25 15.7 15.05 g/L Hematocrit 49.65 44.75 43.25 % Mean corpuscular volume 66.95 66 66.8 fL Mean corpuscular volume hemoglobin 23.3 23.15 23.3 pg Mean corpuscular hemoglobin concentration 34.8 35.1 34.85 g/dL Reticulocytes 69.4 68.25 52.3 109/L 8.79 7.2 8.01 K/uL Leukocytes White blood cells Neutrophils 4.925 3.895 4.915 K/uL Lymphocytes 2.87 2.785 2.475 K/uL Monocytes 0.34 0.215 0.275 K/uL Eosinophils 0445 0.16 0.2 K/uL Basophils 0.165 0.11 0.11 K/uL platelets 239.5 273 274.5 K/uL Mean platelet volume 11.3 11.2 10 fL Prothrombin time 6.7 7.35 6.8 Seconds Activated partial thromboplastin time 12.6 12.3 12.7 Seconds Fibrinogen 291 151.5 201.5 mg/dL Thrombocytes Male and female dogs received indicated doses of α-TEA lysine salt daily for 28 days After 28 daily treatments, blood was collected from dogs per treatment group and analyzed for blood cells and coagulation parameters Guerrouahen et al BMC Cancer (2016) 16:199 Page 10 of 16 Table Mean serum chemistry parameters in dogs 10 days prior to treatment Vehicle Males Females Alanine transaminase (ALT) 100 mg/kg 300 mg/kg 1500 mg/kg Unit Mean SD N Mean SD N Mean SD N Mean SD N 58.2 47.5 55.8 42.2 37.7 5.4 36.8 15.2 U/L Aspartate transaminase (AST) 27.3 2.1 29.8 5.4 32.5 3.8 29.7 3.7 U/L Alkaline phosphatase (ALKP) 91.2 20.5 109.8 39.9 93.8 21.7 128.7 40.1 U/L Lactate deshydrogenase (LDH) 75.3 24.7 71.3 14.4 76 13.8 67.5 24.5 U/L Total bilirubin (TBILI) 0 0 0 0 mg/dL Gamma-Glutamyl transferase (GGT) 0 0 0 0 U/L Creatine phosphokinase (CK) 0 0 0 0 U/L Sodium (Na) 146 1.5 145.5 146.2 1.3 146.7 0.8 mmol/L Potassium (K) 4.65 0.19 4.65 0.17 113.3 113 1.1 mmol/L Chloride (CL) 113.5 1.5 113.3 113 1.1 113.5 2.4 mmol/L Calcium (CA) 10.13 0.19 10.28 0.15 10.43 0.3 10.53 0.45 mg/dL Inorganic phosphorus (PHOS) 5.43 0.77 5.98 0.7 5.23 0.38 5.3 0.45 mg/dL Blood urea nitrogen (BUN) 14.2 0.8 14.5 0.6 17 2.7 16 2.1 mg/dL Creatinine (CREA) 0.63 0.12 0.58 0.05 0.6 0.67 0.08 mg/dL Glucose (GLU) 97.3 3.5 91.3 8.3 92.3 7.4 96 8.9 mg/dL Total protein (TPRO) 4.75 0.24 4.98 0.29 4.9 0.14 4.87 0.26 g/dL Albumin (ALB) 2.88 0.17 2.93 0.26 2.92 0.12 2.83 0.28 g/dL Globulin (GLOB) 1.87 0.18 2.05 0.13 1.98 0.04 2.03 0.23 g/dL Albumin/Globulin ratio (A/G) 1.555 0.165 1.431 0.155 1.471 0.051 1.415 0.249 Ratio Cholesterol (CHOL) 168.3 48.5 160.8 9.3 150.5 31.5 163.8 24 mg/dL Triglycerides (TRIG) 37.3 8.2 38.3 5.9 36 40.2 mg/dL Alanine transaminase (ALT) 31.8 6.6 32.8 7.4 30.3 4.1 37.5 16.3 U/L Aspartate transaminase (AST) 29.3 4.1 31.8 11.2 27.5 3.4 28.3 3.7 U/L Alkaline phosphatase (ALKP) 100 32 83.8 18.3 95.7 25.5 75.3 13.7 U/L Lactate deshydrogenase (LDH) 60.3 18.1 65.3 25.1 52.2 15.4 62.2 21.8 U/L Total bilirubin (TBILI) 0.02 0.04 0 0 0 mg/dL Gamma-Glutamyl transferase (GGT) 0 0 0 0 U/L Creatine phosphokinase (CK) 165.7 37.4 161.5 73.7 141 30.1 142 43.6 U/L Sodium (Na) 146.8 1.5 147.3 1.7 146.7 147.3 0.8 mmol/L Potassium (K) 4.48 0.26 4.48 0.05 4.43 0.31 4.43 0.21 mmol/L Chloride (CL) 114.2 2.1 116 2.2 114.7 2.9 115 1.3 mmol/L Calcium (CA) 10.48 0.26 10.58 0.21 10.95 0.27 10.82 0.31 mg/dL Inorganic phosphorus (PHOS) 5.3 0.23 4.83 0.31 5.35 0.55 5.17 0.5 mg/dL Blood urea nitrogen (BUN) 15.2 2.3 13.3 14.2 2.6 14 mg/dL Creatinine (CREA) 0.57 0.05 0.6 0.58 0.08 0.65 0.08 mg/dL Glucose (GLU) 99.3 99.3 7.5 99.7 7.4 94.5 mg/dL Total protein (TPRO) 4.85 0.27 0.28 4.8 0.14 5.13 0.16 g/dL Albumin (ALB) 2.95 0.26 3.1 0.14 2.95 0.18 3.13 0.16 g/dL Globulin (GLOB) 1.9 0.09 1.9 0.18 1.85 0.14 6 g/dL Albumin/Globulin ratio (A/G) 1.556 0.157 162.3 10.5 1.607 0.21 1.567 0.082 Ratio Cholesterol (CHOL) 160 20.2 162.3 10.5 142.3 11.3 158.2 33 mg/dL Triglycerides (TRIG) 37.3 3.8 36 14.1 34 4.8 46.3 10.4 mg/dL Male and female dogs received indicated doses of α-TEA lysine salt daily for 28 days Ten days before the treatment start, blood was collected from dogs per treatment group (0, 300, 1500 mg/kg) or dogs (100 mg/mL) and analyzed for serum chemistry parameters Guerrouahen et al BMC Cancer (2016) 16:199 Page 11 of 16 Table Mean serum chemistry parameters 29 days after the start date in male dogs Vehicle Males Females Alanine transaminase (ALT) 100 mg/kg 300 mg/kg 1500 mg/kg Unit Mean SD N Mean SD N Mean SD N Mean SD N 33.5 9.9 32 8.6 37.3 6.8 51 25.9 U/L Aspartate transaminase (AST) 34.8 3.2 36.5 5.7 46 8.8 49.8 5.9 U/L Alkaline phosphatase (ALKP) 96 24.4 104.3 37.1 116 71.9 227 315.9 U/L Lactate deshydrogenase (LDH) 57.8 6.4 61 18.7 70 21.8 55 18.9 U/L Total bilirubin (TBILI) 0.03 0.05 0.03 0.05 0.03 0.05 0 mg/dL Gamma-Glutamyl transferase (GGT) 0 0 0 0 U/L Creatine phosphokinase (CK) 246.3 79.8 185.3 61.2 272.3 145.6 133 29.1 U/L Sodium (Na) 148.5 1.3 148 0.8 147.8 0.5 148 1.4 mmol/L Potassium (K) 4.35 0.31 4.5 0.26 4.08 0.32 4.48 0.21 mmol/L Chloride (CL) 115.8 115 0.8 115 1.3 118.8 0.5 mmol/L Calcium (CA) 10.83 0.25 10.88 0.33 11.05 0.13 10.35 0.29 mg/dL Inorganic phosphorus (PHOS) 5.13 0.75 5.45 0.29 4.68 0.26 3.98 0.32 mg/dL Blood urea nitrogen (BUN) 18.3 2.1 18.8 3.3 19.3 1.9 24.3 1.7 mg/dL Creatinine (CREA) 0.6 0.14 0.6 0.58 0.1 0.55 0.08 mg/dL Glucose (GLU) 102.5 4 96.3 96.8 11.2 80.8 8.4 mg/dL Total protein (TPRO) 5.25 0.3 5.38 0.28 5.38 0.24 4.45 0.42 g/dL Albumin (ALB) 3.33 0.26 3.23 0.29 3.18 0.21 2.45 0.1 g/dL Globulin (GLOB) 1.93 0.1 2.15 0.06 2.2 0.14 0.34 g/dL Albumin/Globulin ratio (A/G) 1.729 0.141 1.502 0.152 1.448 0.14 1.247 0.184 Ratio Cholesterol (CHOL) 168.8 35.4 176 28 152.3 19.4 104.5 183.4 mg/dL Triglycerides (TRIG) 34 8.1 41.3 9.7 33.3 5.6 45.5 14.3 mg/dL Alanine transaminase (ALT) 27.3 5.1 26.3 1.9 35.8 10.2 45.5 20.9 U/L Aspartate transaminase (AST) 29 4.7 36.3 5.3 31.5 3.1 45.8 19.5 U/L Alkaline phosphatase (ALKP) 98.8 12.4 78.8 22.5 178 131.5 251.3 211.2 U/L Lactate deshydrogenase (LDH) 82.3 25.8 74 19.8 69.5 5.9 73 19.5 U/L Total bilirubin (TBILI) 0.03 0.05 0.05 0.06 0.03 0.05 0.03 0.05 mg/dL Gamma-Glutamyl transferase (GGT) 0 0.3 0.5 0 0 U/L Creatine phosphokinase (CK) 162.8 54.3 198.8 44.1 162.3 40.6 147.3 47.6 U/L Sodium (Na) 147.8 148.5 1.9 148.5 0.6 148.3 mmol/L Potassium (K) 4.4 0.34 4.53 0.17 4.38 0.31 4.65 0.34 mmol/L Chloride (CL) 114.5 1.7 115.5 115.5 117.8 2.8 mmol/L Calcium (CA) 10.7 0.35 11.05 0.38 11.05 0.37 10.25 0.84 mg/dL Inorganic phosphorus (PHOS) 5.03 0.54 4.25 0.9 4.4 0.47 4.35 0.58 mg/dL Blood urea nitrogen (BUN) 17.5 2.4 14.5 1.3 18.5 1.3 23.5 7.9 mg/dL Creatinine (CREA) 0.5 0.08 0.58 0.1 0.53 0.1 0.63 0.05 mg/dL Glucose (GLU) 103.3 4.8 102.8 11.5 108.8 7.8 84.8 2.1 mg/dL Total protein (TPRO) 5.28 0.17 5.35 0.31 5.3 0.22 4.65 0.3 g/dL Albumin (ALB) 3.35 0.17 3.38 0.25 3.35 0.19 2.8 0.22 g/dL Globulin (GLOB) 1.93 0.1 1.98 0.1 1.95 0.17 1.85 0.13 g/dL Albumin/Globulin ration (A/G) 1.745 0.142 1.71 0.109 1.729 0.191 1.516 0.118 Ratio Cholesterol (CHOL) 167.5 24.6 179 22.4 147.3 10.6 98.3 13 mg/dL Triglycerides (TRIG) 40 9.8 42 8.8 43.5 5.8 50 8.3 mg/dL Male dogs received indicated doses of α-TEA lysine salt daily for 28 days After 28 daily treatments, blood was collected from dogs per treatment group and analyzed Guerrouahen et al BMC Cancer (2016) 16:199 Page 12 of 16 Table Mean serum chemistry parameters in dogs at the end of the recovery period (Day 57) Vehicle Males Females Alanine transaminase (ALT) 300 mg/kg 1500 mg/kg Unit Mean N Mean N Mean N 33 36.5 33 U/L Aspartate transaminase (AST) 31 30.5 33 U/L Alkaline phosphatase (ALKP) 81.5 66 95 U/L Lactate deshydrogenase (LDH) 36 50 45.5 U/L Total bilirubin (TBILI) 0.05 0.05 0.1 mg/dl Gamma-Glutamyl transferase (GGT) 2 U/L Creatine phosphokinase (CK) 119 142.5 169 U/L Sodium (Na) 147.5 146.5 148.5 mmol/L Potassium (K) 4.3 4.6 4.4 mmol/L Chloride (CL) 116 115 116.5 mmol/L Calcium (CA) 10.65 10.45 10.55 mg/dL Inorganic phosphorus (PHOS) 5.15 5.1 5.3 mg/dL Blood urea nitrogen (BUN) 13.5 15.5 21 mg/dL Creatinine (CREA) 0.65 0.6 0.7 mg/dL Glucose (GLU) 99 99.5 96 mg/dL Total protein (TPRO) 5.3 5.15 5.4 g/dL Albumin (ALB) 2.9 2.95 g/dL Globulin (GLOB) 2.3 2.25 2.45 g/dL Albumin/Globulin ratio (A/G) 1.314 1.289 1.219 Ratio Cholesterol (CHOL) 149 154 163.5 mg/dL Triglycerides (TRIG) 24 24 41 mg/dL Alanine transaminase (ALT) 22.5 31 27.5 U/L Aspartate transaminase (AST) 33 29 26.5 U/L Alkaline phosphatase (ALKP) 73 63.5 50 U/L Lactate deshydrogenase (LDH) 52 49.5 47 U/L Total bilirubin (TBILI) 0.1 0.1 0.1 mg/dL Gamma-Glutamyl transferase (GGT) 2 0 U/L Creatine phosphokinase (CK) 154.5 146 120 U/L Sodium (Na) 146.5 146.5 147 mmol/L Potassium (K) 4.5 4.5 4.1 mmol/L Chloride (CL) 115.5 115.5 116.5 mmol/L Calcium (CA) 10.95 10.65 10.5 mg/dL Inorganic phosphorus (PHOS) 5.15 4.95 4.5 mg/dL Blood urea nitrogen (BUN) 15.5 13.5 14.5 mg/dL Creatinine (CREA) 0.65 0.7 0.65 mg/dL Glucose (GLU) 103.5 101.5 100 mg/dL Total protein (TPRO) 5.7 5.15 5.25 g/dL Albumin (ALB) 3.3 3.1 g/dL Globulin (GLOB) 2.4 2.15 2.15 g/dL Albumin/Globulin ratio (A/G) 1.377 1.395 1.444 Ratio Cholesterol (CHOL) 190.5 152 161 mg/dL Triglycerides (TRIG) 29.5 30.5 32 mg/dL Male and female dogs received indicated doses of α-TEA lysine salt daily for 28 days At the end of the recovery period, on day 57, blood was collected from dogs per treatment group and analyzed Guerrouahen et al BMC Cancer (2016) 16:199 Page 13 of 16 (BUN), and mean aspartate aminotransferase activity (AST) values for the 1500 mg/kg males group were statistically higher compared to the controls Although not statistically different, the mean CL, mean BUN, and mean AST values for the 1500 mg/kg females were minimally higher compared to the controls The mean total protein concentration (TPRO), mean albumin (ALB) concentration, mean albumin to globulin ratio (A/G), and mean cholesterol (CHOL) concentration values for the 1500 mg/kg females were statistically lower compared to the controls Although not shown to be statistically different, the mean glucose concentration (GLU) value for the 1500 mg/kg females group was lower compared to the controls (Tables and 7) At Day 57 (Table 8), the mean BUN concentration value for the 300 mg/kg and 1500 mg/kg males groups was higher compared to the controls This difference was attributed to the previous anorexia seen in the 1500 mg/kg males group, but was not of such a magnitude as to be considered an indication of renal toxicity No urinalysis changes were reported at the highest dose (1500 mg/kg) 1500 mg/kg males and 1500 mg/kg females), kidney, liver (1500 mg/kg, male), and thymus (300 and 1500 mg/ kg males and 100 and 1500 mg/kg females) These findings were considered to be incidental No α-TEA related macroscopic changes were observed at the end of dose administration or recovery phase, and there were no changes in organ weight Interestingly, α-TEA-related histomorphologic changes were noted at 1500 mg/kg on Day 29 in the duodenum (mild, diffuse villous atrophy) at 1500 mg/ kg, jejunum (minimal, focal or multifocal dilation of a crypt) at all dose levels, kidney (mild, diffuse, bilateral vacuolation of the epithelium lining the proximal tubules) at 1500 mg/kg, and liver (minimal, multifocal hepatocellular necrosis with minimal, multifocal sub-acute inflammation and moderate, diffuse vacuolation of hepatocytes) at 1500 mg/kg At the end of recovery period, minimal focal dilation of jejunal crypt and moderate diffuse vacuolation of hepatocytes was present at 1500 mg/kg and mild diffuse depletion of lymphocytes of the thymic cortex was noted at 300 mg/kg Histology of major organs Toxicokinetics parameters Dogs were euthanized the day following the end of dosing period or following a 28-day recovery period (day 57) by an overdose of sodium pentobarbital followed by exsanguination A necropsy with tissue collection was conducted and included examination of the carcass and muscular/skeletal system, orifices, cranial cavity and external surface of the brain, neck, thoracic, abdominal and pelvic cavities associated with their organs There were few and minimal α-TEA-related histomorphologic or organ weight changes observed at the end of dose administration in the adrenal glands (1500 mg/kg females), duodenum (1500 mg/kg males), jejunum (100 and Samples of blood were collected from the jugular vein of all animals at 1, 2, 4, 8, 24 h at day and day 28 when animals received the test article The validated LC-MS/MS method was used to analyze plasma α-TEA concentration Mean plasma α-TEA concentrations in all samples obtained at h post-dose from control animals on Day were below the lower limit of quantitation (50.0 ng/mL), indicating no exposure of treated animals to α-TEA prior to dosing As shown in Table 9, the mean Tmax ranged from 16.0 to 24.0 h on Day and from 6.3 to 10.7 h on Day 28 Elimination half-life values were not calculated or not reported on Days and 28 due to insufficient data Table Mean toxicokinetic parameters at day and 28-day dosing of α-TEA lysine salt Day Group α-TEA Dose (mg/kg) Sex Tmax (hr) T1/2 (hr) Cmax (ng/mL) AUC0-24 (hr*ng/ml) 100 Male 24 NC 20200 335000 100 Female 20 NC 23900 35100 300 Male 16 NC 41000 718000 300 Female 18.7 NC 48600 72900 1500 Male 24 NC 69100 1170000 1500 Female 16 NC 76900 1240000 28 100 Male 6.3 NC 148000 3290000 28 100 Female NC 16000 3620000 28 300 Male 7.3 NC 211000 4740000 28 300 Female NC 212000 4670000 29 1500 Male NC 206000 4580000 28 1500 Female 10.7 NC 192000 4210000 Blood were collected collected at 1, 2, 4, 8, and 24 h following the first α-TEA administration and 2, 8, 24, 72, and 168 h following the last administration (day 28) The validated method (high performance liquid chromatography with mass spectrometric detection) was used to analyse plasma α-TEA concentration Cmax, Tmax, T1/2, AUC were determined using WinNonlin Version 6.2 software system Guerrouahen et al BMC Cancer (2016) 16:199 points for the elimination phase On Days and 28, increases in Cmax and AUC0-24 were less than proportional for both males and females On the first day of administration, for a 15-fold increase in dose from 100 to 1500 mg/ kg, Cmax values increased 3.42-fold for males and 3.22-fold for females and AUC0-24 values increased 3.49-fold for males and 3.53-fold for females On Day 28, α-TEA exposure was similar at 300 and 1500 mg/kg, this reflects a saturation phenomenon at 300 mg/kg dose level and above Page 14 of 16 For a 3-fold increase in dose from 100 to 300 mg/kg, Cmax increased 1.43-fold for males and 1.33-fold for females and AUC0-24 increased 1.44-fold for males and 1.29-fold for females Cmax and AUC0-24 values were similar between males and females The ratio of Cmax for males to Cmax for females at each dose level ranged from 0.84 to 0.90 on Day and from 0.93 to 1.07 on Day 28 The ratio of AUC0-24 for males to AUC0-24 for females at each dose level ranged from 0.94 to 0.98 on Day and from 0.91 to Fig Mean plasma α-TEA lysine salt concentration versus time at day for male and female dogs Male dogs received indicated doses of α-TEA lysine salt on day Plots of α-TEA plasma concentration versus time are presented Guerrouahen et al BMC Cancer (2016) 16:199 1.09 on Day 28 The AUC ratios for α-TEA LS (ratio of AUC0-24 on Day 28 to the AUC0-24 on Day 1) was 9.82 for males and 10.31 for females at 100 mg/kg), 6.60 for males and 6.41 for females at 300 mg/kg, and 3.91 for males and 3.40 for females at 1500 mg/kg Plots of α-TEA plasma concentration versus time are presented in Figs and for Day and Day 28, respectively Exposure of animals on Day 28 was higher than exposure on Day 1, indicating accumulation of α-TEA with multiple dosing The extent of accumulation was greatest at the low-dose level and Page 15 of 16 decreased with increasing dose, presumably due to saturation of exposure at the mid- and high-dose levels No sex-specific differences in toxicokinetics values could be reported Discussion This toxicology and pharmacokinetics study of GMP grade α-TEA was conducted to support an Investigator New Drug (IND) application to the FDA to conduct a first-in-human clinical trial in patients with advanced Fig Mean plasma α-TEA lysine salt concentration versus time at day 28 for male and female dogs Female dogs received indicated doses of α-TEA lysine salt on day Plots of α-TEA plasma concentration versus time are presented Guerrouahen et al BMC Cancer (2016) 16:199 cancer based on the immunological and pro-apoptotic properties of α-TEA Our results demonstrate that orally administered α-TEA LS reduces tumor growth and increases overall survival in mice bearing established tumors This anti-tumor activity is comparable to that of α-TEA free acid that we previously reported [11] However, unlike α-TEA free acid, which is difficult to manufacture for human use due to its liquid crystal properties, α-TEA LS is crystalline in nature and can be delivered in aqueous form to animals In the non-rodent dose escalation study, in which optimal conditions and timing of orally administered α-TEA LS were assessed, there were no signs of toxicity in dogs that received daily doses of up to 1500 mg/kg No moribundity or mortality was observed at any of the dose levels tested throughout the treatment period No genderrelated differences were observed Minor histomorphologic alterations were observed at the highest dose but these changes were mostly reversed at the end of the recovery period Clinical pathology changes were limited to increased white blood cell (lymphocyte and neutrophil) counts, and fibrinogen levels at 1500 mg/kg; these changes were likely related to mild inflammation and/or stress but all these findings were resolved by the end of recovery period Although a full and complete toxicokinetic profile could not be determined from the study, there was an apparent less than dose proportional increase in AUC over the duration of the study The extent of accumulation was greatest at the low-dose level and decreased with increasing dose, presumably due to saturation of exposure at the mid- and high-dose levels Conclusion In summary, in this comprehensive examination of the prolonged use of GMP-grade α-TEA LS in dogs, followed by a 28-day recovery period, we report absence of any dose limiting toxicity Our findings suggest that α-TEA is safe and tolerated and determined that the highest nonseverely toxic dose (HNSTD) to be 1500 mg/kg These findings formed the basis for initiating a first-in-human clinical trial of α-TEA in patients with advanced cancer, which is ongoing Abbreviations α-TEA: Alpha-Tocopheryloxyacetic free acid; α-TEA LS: α-TEA Lysine Salt; HPLC: High Performance Liquid Chromatography Page 16 of 16 Acknowledgements This study was supported by funds from The Safeway Foundation and Providence Cancer Center Received: 10 October 2015 Accepted: 28 February 2016 References Lawson KA, Anderson K, Menchaca M, Atkinson J, Sun L, Knight V, Gilbert BE, Conti C, Sanders BG, Kline K Novel vitamin E analogue decreases syngeneic mouse mammary tumor burden and reduces lung metastasis Mol Cancer Ther 2003;2(5):437–44 Neuzil J, Dong LF, Ramanathapuram L, 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breast cancer Mol Cancer Ther 2009;8(6):1570–8 11 Hahn T, Akporiaye ET Repeat dose study of the novel proapoptotic chemotherapeutic agent alpha-tocopheryloxy acetic acid in mice Anticancer Drugs 2012;23(4):455–64 Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to find the most relevant journal Competing interests The authors declare that they have no competing interests Authors’ contributions ETA, BC, WU, conception and designed research; TH planned research, ZA performed research; ETA, BC, WU analyzed data; BSG wrote the manuscript with contribution from the coauthors All authors read and approved the final manuscript • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit ... Fig Mean plasma α-TEA lysine salt concentration versus time at day 28 for male and female dogs Female dogs received indicated doses of α-TEA lysine salt on day Plots of α-TEA plasma concentration... findings formed the basis for initiating a first -in- human clinical trial of α-TEA in patients with advanced cancer, which is ongoing Abbreviations α-TEA: Alpha-Tocopheryloxyacetic free acid; α-TEA. .. h at day and day 28 when animals received the test article The validated LC-MS/MS method was used to analyze plasma α-TEA concentration Mean plasma α-TEA concentrations in all samples obtained